Breast cancer arises from estrogen-responsive breast epithelial cells. Estrogen activity is thought to promote the development of breast cancer, and many breast cancers are initially dependent on estrogen at the time of diagnosis. Anti-estrogen compositions have therefore been used to treat breast cancer.
A frequent mechanism of increased gene expression in human cancers is amplification, i.e., the copy number of a DNA sequence is increased, in a cancer cell compared to a non-cancerous cell. In breast cancer, commonly amplified regions are derived from 17q21, 8q24, and 11q13 which encode erbB-2, c-myc, and cyclic D1 respectively (Devilee et al., 1994, Crit. Rev. Oncog. 5:247-270). Recently, molecular cytogenetic studies have revealed the occurrence in breast cancers of additional regions of increased DNA copy number (Isola et al., Am. J. Pathol. 147:905-911, 1995; Kallioniemi et al., Proc. Nati. Acad. Sci. USA 91:2156-2160, 1994; Muleris et al., Genes Chromo. Cancer 10:160-170, 1994; Tanner et al., Cancer Research 54:4257-4260, 1994; Guan et al., Nat. Genet. 8:155-161, 1994).
Breast cancer is the second leading cause of cancer deaths in American women, and it is estimated that an American woman has at least a 10% cumulative lifetime risk of developing this disease. Early diagnosis is an important factor in breast cancer prognosis and affects not only survival rate, but the range of therapeutic options available to the patient. For instance, if diagnosed early, a xe2x80x9clumpectomyxe2x80x9d may be performed, whereas later diagnosis tends to be associated with more invasive and traumatic surgical treatments such as radical mastectomy. The treatment of other cancers likewise is benefitted by early diagnosis, for instance the prognosis in the treatment of lung cancer, colorectal cancer and prostate cancers is greatly improved by early diagnosis. There is a need for a simple and reliable method of diagnosis of cancers in general and of breast cancer in particular. There is a need for a method of screening for compounds that inhibit the interaction between an estrogen receptor ER and an ER-dependent nuclear receptor co-activator molecule in order to identify molecules useful in research diagnosis and treatment of cancer. There is also a need for a method for identifying tamoxifen-sensitive cancer patients in order to better manage treatment. A solution to these needs would improve cancer treatment and research and would save lives.
The inventors have discovered that the AIB1 protein (Amplified In Breast Cancer-1) is a member of the Steroid Receptor Coactivator-1 (SRC-1) family of nuclear receptor co-activators that interacts with estrogen receptors (ER) to enhance ER-dependent transcription. The inventors have further discovered that the AIB1 gene is amplified and over-expressed in certain cancers including breast cancer, and that detection of amplified AIB1 genes can therefore be used to detect cancerous cells. Importantly, the inventors have also found that AIB1 amplification is not confined to breast cancer but is also found in cancers of the lung, ovary, head and neck, colon, testicles, bladder, prostate, endometrium, kidney, stomach and also in pheochromocytoma, melanoma, ductal carcinoma and carcinoid tumor. Such a finding means that AIB1 may be useful in the detection and treatment of all of the aforementioned cancers which include some of the most prevalent and deadly diseases in the western world.
The inventors have also discovered that AIB1 interacts with the proteins p300 and CBP, which are nuclear cofactors that interact with other nuclear factors to promote transcription (Chacravarti et al., Nature (383) 99-103 1996; Lundblad et al., Nature (374) 85-88 1995). The inventors have, furthermore, determined that in cells with stable over-expression of AIB1, there is a dramatic increase in steroid receptor activation (almost a 100-fold increase) leading to a corresponding increase in transcriptional activation. The inventors have also used monoclonal anti-AIB1 antibodies to demonstrate that AIB1 gene amplification is directly correlated with increased AIB1 expression, and that these amplified copies of the gene are expressed in physiological conditions. The inventors have found that AIB1 is the human ortholog of the mouse ER-dependent transcriptional activator p/CIP, with the proteins having an overall amino acid identity of 81.6%. These finding support the physiological role for AIB1 in cancer cells as a cofactor involved in transcriptional regulation.
The invention features a substantially pure DNA which includes a sequence encoding an AIB1 polypeptide, e.g., a human AIB1 polypeptide, or a fragment thereof. The DNA may have the sequence of all or part of the naturally-occurring AIB1-encoding DNA or a degenerate variant thereof. AIB1-encoding DNA may be operably linked to regulatory sequences for expression of the polypeptide. A cell containing AIB1 encoding DNA is also within the invention.
The invention also includes a substantially pure DNA containing a polynucleotides which hybridizes at high stringency to a AIB1-encoding DNA or the complement thereof. A substantially pure DNA containing a nucleotide sequence having at least 50% sequence identity to the full length AIB1 CDNA, e.g., a nucleotide sequence encoding a polypeptide having the biological activity of a AIB1 polypeptide, is also included.
The invention also features a substantially pure human AIB1 polypeptide and variants thereof, e.g., polypeptides with conservative amino acid substitutions or polypeptides with conservative or non-conservative amino acid substitutions which retain the biological activity of naturally-occurring AIB1.
Diagnostic methods, e.g., to idertify cells which harbor an abnormal copy number of the AIB1 DNA, arc also encompassed by the invention. An abnormal copy number, e.g., greater than the normal diploid copy number, of AIB1 DNA is indicative of an aberrantly proliferating cell, e.g., a steroid hormone-responsive cancer cell.
The invention also includes antibodies, e.g., a monoclonal antibody or polyclonal antisera, which bind specifically to AIB1 and can be used to detect the level of expression of AIB1 in a cell or tissue sample. An increase in the level of expression of AIB1 in a patient-derived tissue sample compared to the level in normal control tissue indicates the presence of a cell proliferative disorder such as cancer.
Screening methods to identify compounds which inhibit an interaction of AIB1 with a steroid hormone receptor, thus disrupting a signal transduction pathway which leads to aberrant cell proliferation, is also within the invention. Proliferation of a cancer cell can therefore be reduced by administering to an individual, e.g., a patient diagnosed with a steroid-responsive cancer, a compound which inhibits expression of AIB1.
The invention also includes a knockout mutant, for example a mouse (or other mammal) from which at least one AIB1 gene has been selectively deleted from its genome. Such a mouse is useful in research, for instance, the phenotype gives insight into the physiological role of the deleted gene. For instance the mutant may be defective in specific biochemical pathways; such a knockout mutant may be used in complementation experiments to determine the role of other genes and proteins to determine if any such genes or proteins complement for the deleted gene. Homozygous and heterozygous mutants are included in this aspect of the invention.
The present invention also includes a mutant organism, for example a mammal such as a mouse which contains more than the normal number of AIB1 genes in its genome. Such a mouse may contain additional copies of the AIB1 gene integrated into its chromosomes, for instance in the form of a pro-virus, or may carry additional copies on extra-chromosomal elements such as plasmids. Such a mutant mouse is useful for research purposes, to elucidate the physiological or pathological role of AIB1. For instance, the role of AIB1 expression as cause or effect in cancers may be investigated by including or transplanting tumors into such mutants, and comparing such mutants with normal mice having the same cancer.
The present invention also includes a mutant organism, for example a mammal, e.g. a mouse, that contains, either integrated into a chromosome or on a plasmid, at least one copy of the AIB1 gene driven by a non-native promoter. Such a promoter may be constitutive or may be inducible. For instance, the AIB1 gene may be operatively linked to a mouse mammary tumor virus (MMTV) promoter or other promoter from a mammalian virus allowing manipulation of AIB1 expression. Such a mutant would be useful for research purposes to determine the physiological or pathological role of AIB1. For instance, over or under expression could be affected and physiological effects observed.
The invention also includes methods for treatment of cancers that involve functions of or alterations in the signaling pathways that use p300 and/or CBP as signal transducing molecules. The treatments of the invention involve targeting of the AIB1 protein or AIB1 gene to enhance or reduce interaction with p300 and/or CBP proteins. For instance, the AIB1 gene sequence as disclosed herein may be used to construct an anti-sense nucleotide. An anti-sense RNA may be constructed that is anti-parallel and complementary to the AIB1 transcript (or part thereof) and which will therefore form an RNA-RNA duplex with the AIB1 transcript, preventing transcription and expression of AIB1. Alternatively, treatments may comprise contacting an AIB1 protein with a molecule that specifically binds to the AIB1 molecule in vivo, thereby interfering with AIB1 binding with other factors such as p300 or CBP. Such processes are designed to inhibit signal transduction pathways involving AIB1, p300, CBP and other factors and therefore inhibit cancer cell proliferation that is effected via these pathways. As explained in more detail below, AIB1 overexpression results in increased ER-dependent transcriptional activity which confers a growth advantage upon AIB1 amplification-bearing clones during the development and progression of estrogen-dependent cancers.
Compounds which inhibit or disrupt the interaction of an AIB1 gene product with a steroid hormone receptor, e.g., ER, are useful as anti-neoplastic agents for the treatment of patients suffering from steroid hormone-responsive cancers such as breast cancer, ovarian cancer, prostate cancer, and colon cancer.
AIB1 polypeptides or peptide mimetics of such polypeptides, e.g., those contaig domains which interact with steroid hormone receptors, can be administered to patients to block the interaction of endogenous intracellular AIB1 and a steroid hormone receptor, e.g., ER in an aberrantly proliferating cell. It is likely that AIB1 interacts with a wide range of human transcriptional factors and that regulation of such interactions will have important therapeutic applications.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The nucleic acid and amino acid sequences listed in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases and three-letter code for amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.
SEQ ID NO:1 shows the nucleic acid sequence of AIB1 cDNA and the corresponding amino acid sequence.
SEQ ID NO:2 shows the amino acid sequence of the Per/Amt/Sim (PAS) domain of AIB1.
SEQ ID NO:3 shows the amino acid sequence of the basic helix-loop-helix domain (bHLH) of AIB1.
SEQ ID NO:4 shows the amino acid sequence of the human AIB1 protein.
SEQ ID NO:5 shows the nucleic acid sequence of primer N8F1.
SEQ ID NO:6 show the nucleic acid sequence of the forward primer designed from the 5xe2x80x2 sequence of pCMVSPORT-B11, PM-U2.
SEQ ID NO:7 shows the nucleic acid sequence of the reverse primer designed from the 5xe2x80x2 sequence of pCMVSPORT-B11, PM-U2.
SEQ ID NO:8 shows the amino acid sequence of the ER-interacting domain of AIB1.
SEQ ID NO:9 shows the nucleic acid sequence of pCIP, the mouse ortholog of AIB1 and the amino acid sequence for this gene.
SEQ ID NO:10 shows the nucleic acid sequence of the forward primer AIB1/mESTF1 used to screen mouse BAC.
SEQ ID NO:11 shows the nucleic acid sequence of the reverse primer AIB1/mESTF1 used to screen mouse BAC.
SEQ ID NO:12 shows the amino acid sequence of pCIP, the mouse ortholog of AIB1.